PROJECT SUMMARY The ability for clinicians to effectively treat bacterial infections with targeted antibacterials in the acute-care settings hinges on diagnostics capable of identifying the pathogen broadly and determining its susceptibility to antibacterials in a timely manner. Bloodstream infection (BSI) is a particularly representative disease because it is the leading cause of death due to infections with rapid disease progression. Unfortunately, the inconvenient delay of blood culture for definitive diagnosis contributes to widespread empiric use of broad- spectrum antibacterials and emergence of multi-drug-resistant pathogens. Toward addressing this critical unmet need, we propose to develop a new molecular diagnostic platform that integrates bacterial detection, species identification (ID), and antibacterial susceptibility testing (AST) from blood samples in a streamlined test. The expected sample-to-answer turnaround time is 90 min for ID and as early as 2-3 hr for AST. Such integrated diagnostic solution within the proposed timeframe will transform acute-care clinicians? ability to establish diagnosis of bacterial infections, need for infection control, and antibacterial treatment based on objective data to improve clinical outcome. Using an innovative microfluidic digital array chip for assaying single cells as a backbone technology, we propose to develop a new molecular diagnostic platform which promises rapid ID and AST and allows customizable workflow and assay tailored to the clinical scenario while adjustable based on real-time results. The array chip seamlessly integrates digitization of cells, brief incubation (under various drug conditions), single-cell PCR (scPCR) or reverse transcriptase PCR (scRT-PCR) and single-cell high-resolution melt (scHRM). Thereby, bacterial pathogen can be detected at the level of single-cells, identified based on species- specific melt curves, and their antibacterial susceptibility profile subsequently assessed by measuring changes in rRNA level as a biosynthetic marker of cell viability. ScPCR/scRT-PCR enables sensitive detection and absolute quantification of rRNA of individual cells critical to rapid and reliable differentiation between viable and no-viable cells; while scHRM overcomes a key limitation of bulk HRM to resolve multiple species for diagnosing polymicrobial infections or discarding contaminations. Since both ID and AST do not rely on culture, they reduce total turnaround time from days to minutes/hours. We have assembled a superb team of multi-disciplinary investigators and industry advisors with complementary expertise and strong track record of team science. We propose the following aims:1) to develop a streamlined BSI diagnostic protocol for integrated ID and AST; 2) to develop a microfluidic array chip that enables ID and AST with single-cell resolution; 3) to develop instrument and analysis programs for single- cell ID and AST; and 4) to demonstrate the single-cell diagnostic platform, we will perform analytical and pilot clinical validation studies.